JP2002179563A - Compositions for inhibiting diseases associated with oxidative damage - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapeutic compound, a composition and a method of using the same for inhibiting a disease associated with oxidative damage. A composition for the treatment or prevention of dysfunction and disease states resulting from oxidative damage, comprising a pharmaceutically acceptable carrier and an active ingredient for administration to a patient, wherein the active ingredient is , Hydroxy PBNs, PBN
A composition which is a compound including a compound selected from the group consisting of esters, acetoxy PBNs, alkyl PBNs, alkoxy PBNs, and phenyl PBNs.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to spin-trapping agents for the treatment of stroke, aging-related dysfunctions and other conditions resulting from oxidative damage, compositions containing them and methods of using them. About.

[0002]

BACKGROUND OF THE INVENTION Oxidized tissue suffers damage, often resulting in permanent damage when it becomes ischemic and then reperfused. Oxygen free radicals are closely related to this damage. The brain is particularly susceptible to ischemia / reperfusion injury, and neurons are more sensitive than glial cells. Certain areas of the brain, such as the hippocampus and spinal cord, are more sensitive than other areas of the brain. As a result, ischemic / reperfusion injury of the brain can have multiple effects, because proper functioning requires complete integrity of all regions.

Several mechanisms can cause ischemic brain damage. For example, mechanisms that respond to selective neuronal damage include glial cell swelling and extensive cerebral edema,
And may differ from a third set of symptom mechanisms that can cause global dysfunction such as seizures. Siesjo
Classified in the Critical Care Med. 16, 954-963 (1988) a number of observations on brain injury into four possible mechanisms. These include A) calcium-mediated cell death, B) protoxic damage (glutamate accumulation), C) free radical cases, and D) acidosis. It appears that all four basic mechanisms are interrelated or collectively contributing to the observed damage. Regarding the free radical mechanism of brain injury, Siesjo concludes that the evidence supporting the involvement of the free radical mechanism is not conclusive.

[0004] Free radicals have been identified as mediators of reperfusion injury. Superoxide (O ^-2) or hydroxyl (OH ^-) The main production site of species such radicals, the mitochondrial respiratory chain, and a sequence catalyzed cyclooxygenase and lipoxygenase. However, radicals are also formed during the autoxidation of many compounds (eg, catecholamines). In some ischemic cases, free radical formation occurs rapidly, for example, causing oxidation of polyene free fatty acids, release and reabsorption of catecholamines, and oxidation of hypoxanthine by xanthine oxidase. Although all of these cases occur during recirculation, when the supply of O ₂ is restored, agonist - shows receptor interactions, energy loss, and / or calcium influx during the insult period, the metabolic cascade reaction caused by . Free radical formation is thought to be the cause of ischemic damage, but such formation can occur and / or Curran et al . , Mol. Cell. Biol. 5, 167-172 (1985).
It was difficult to directly demonstrate that it could be concluded that it was sufficient to overwhelm the antioxidant defenses of a tissue as described in US Pat. However, recently, evidence has been obtained that ischemia can form conjugated dienes and that malondialdehyde accumulates in tissues. Nevertheless, free radical damage to unsaturated acyl chains, proteins or nucleic acids in phospholipids has not been conclusively shown to play an important role in ischemic necrosis. At this time, there is relatively strong evidence that free radical mechanisms are involved in vascular injury and relatively weak evidence of damage affecting nerves and glial cells.

[0005] Free Radical Bi from Hall and Braughler
ol. Med. 6, 303-313 (1989), Kontos, Taylor, Matal.
on and Ward editing, Physiology of Oxygen Radicals , p
p. 207-216 (Am. Physiol. Soc. Bethesda, MD 1986),
And Ernster's Critical Care Med. 16, 947-953
(1988), et al., Show enormous amounts of evidence associated with oxidative head injury and spinal cord injury, and hemorrhagic and ischemic stroke. Most studies closely related to the main role of oxidative damage in ischemic / reperfusion injury of the head fall into one of two types of studies: the use of reagents to prevent oxidative cases. Studies showing protection by addition or studies designed to observe free radicals.

[0006] Although there are currently no drugs approved for clinical use to treat tissue damage due to ischemia, several compounds have been proposed to be potentially effective. Mannitol, an oxygen scavenger, was added to the reperfusion solvent to reduce damage to the tissue for transplantation. Superoxide dismutase (SOD) has been suggested as a means of suppressing oxidative damage in vivo. The most promising compounds that interfere with peroxidant generation are JM McCall, Acta
Anesthesia Belgica , First Antwerp Int. Trauma Sym
p., modified prednisone (prednison)
lazaroides, which have been reported to be effective when administered during or after ischemia. See, for example, Braughler et al . , J. Biol. Chem. 262, 104 .
38-10440 (1987), a 21-amino acid steroid (474006F), known to block peroxidation of brain homogenates, induced gerbils in three hours of unilateral carotid artery occlusion. Hall et al. Showed protection from injury in Stroke 19, 997-1002 (1988). White and
Adv. Free Radical Biol. Me by Aust and co-workers
d. 1, 1-17 (1985) and Babbs, Resuscitation 1
3, 165-173 (1986) states that iron chelating agents
It has been shown to protect against reperfusion injury.

For direct evidence of oxidative events during ischemic / reperfusion injury of the brain, appropriate observations have been made using spin trapping, salicylic acid hydroxylation, protein oxidation, and oxidative damage of nucleic acids. . Spin trapping has provided clear evidence closely linked to free radical production during ischemia / reperfusion injury. Incubate with rat brain homogenate exposed to very low oxygen tension for 5 minutes as described in Neurological Res. 8, 214-220 (1986), Imaizumi et al . It was clearly shown that it is possible to trap lipid-type radicals. Kirsch
Et al . , In the preface of Pediatric Res. 21, 202A (1987), state that PBN trapped free radicals in the brain after ischemia from animals pretreated with spin trap. McCay's group includes Arch. Biochem. Biophys. 24
4, as described in 156-160 (1986), a free radical spin trapping in mouse brain, and, J. C
As described in lin. Invest. 82, 476-485 (1988), free radicals spin trapped by PBN were found in blood from intact dog hearts recovered from "stunned" myocardium. Showed that it exists. As described, the spin-trapped free radical is
Apparently free radicals of the lipid type, appearing within 1 minute after release of the occlusion and peaking in intensity about 5 minutes after the start of reperfusion. PBN facilitates post-ischemic functional recovery of a "collapsed" heart.

Although PBN has been used in many research studies, data can support that PBN or any other spin-trapping agent can be useful in vivo, especially for the treatment of tissue damage in the central nervous system. There is no. In vivo, the drug (1) crosses the blood-brain barrier,
(2) It must be able to act to reduce tissue damage during or after the ischemic period.

Age-related changes in central nervous system function include:
It is commonly associated with cell loss, dilatation of the lateral ventricle, and short-term memory loss. There is no generally accepted precise mechanism for functional changes that occur as a result of aging or other age-related diseases. Several mechanisms have been proposed for the production of oxidants in the brain. In particular, among transition metals, iron and copper have been suggested as mediators of this oxidation. With increased protein oxidation, there is a marked decrease in the receptor system of certain neurotransmitters. For example, decreased muscarinic receptors and other acetylcholine systems have been characterized as being associated with altered function in Alzheimer's disease. It is also hypothesized that aging is associated with multiple short-term ischemia (multiple infarct states or transient ischemic attacks) that can result in the production of oxidized proteins over that period.

[0010] Changes associated with ischemic brain disease include changes in calcium distribution, increased release of irritable neurotransmitters,
It has also been proposed to be the result of the production of free radicals and the acidosis that naturally results from an increase in weakly bound metals in cells that catalyze the generation of oxygen free radicals. These changes are mainly restricted to nerve cells. However, reactive glial cells have been shown to appear mainly after neuronal damage.

[0011] Treatment of senile dementia has been severely limited due to the difficulty in developing a suitable model for studying this condition. This is due to the fact that aging is a very complex condition that is difficult to model due to the lack of certain information, especially related to the functional and biochemical basis associated with human senile dementia. Is due. The use of animal models may be associated with a truly functional senescence when the brain is not really aging, or when using aging animals, or in some cases, despite little knowledge of the origin of aging. It depends heavily on the model system used for brain observation, such as when it is impossible to prove it.

[0012] With respect to aging, elevated levels of oxidized proteins in tissues have been shown in various systems, both nervous and non-nervous, indicating that aging in the central nervous system. Age-related dysfunction suggests that it may be related to the accumulation of oxidized proteins and oxidized macromolecules in nerve cells throughout the central nervous system. The hypothesis is that cells that have accumulated oxidized proteins have reduced function and their ability to maintain their specific role in the specific location of the central nervous system in which they reside. Things. This hypothesis has been suggested by several investigators, but there is no practical study providing and altering oxidized proteins in the central nervous system and correlating functional changes in animal organs. If truly linked to brain dysfunction, such an approach could provide a basis for regenerating age-related, viable but deficient neuronal cells. .
Such an approach is performed on cells with reduced function and viability.

[0013]

Accordingly, one object of the present invention is to provide compounds, compositions and compositions thereof useful for inhibiting or ameliorating ischemic damage in vivo, especially in the CNS, spinal cord and eyes. The purpose is to provide usage.

[0014] It is a further object of the present invention to provide compositions and methods of use thereof that are useful in vivo to prevent or ameliorate free radical damage from infection and inflammation.

It is a still further object of the present invention to provide a composition for use in inhibiting energy loss of cells during ischemia and methods of use thereof.

Yet another object of the present invention is to provide, in addition to dysfunction from ischemia, progressive neuronal cell loss from disease or drugs and alcohol abuse, photo-oxidation and exposure to high pressure or oxygen. It is an object of the present invention to provide compositions useful for treating various dysfunctions, including dysfunctions derived therefrom, and methods of using the same.

[0017] It is another object of the present invention to provide compositions and methods of use thereof that are useful in preventing or ameliorating age-related functional deficits.

It is a further object of the present invention to provide compositions and methods of use that are useful for preventing or ameliorating cognitive deficits associated with infection or inflammation.

It is a still further object of the present invention to provide compositions and methods of use thereof that reduce cognitive dysfunction after trauma.

[0020]

SUMMARY OF THE INVENTION As a compound which is an active ingredient for treating or inhibiting stroke or other symptoms related to ischemic damage, aging or other oxidative tissue damage, a spin trapping agent, preferably Discloses α-phenyl-t-butyl nitrone (PBN) or a spin trapping derivative thereof, and compositions comprising them in a pharmaceutically acceptable carrier for administration to a patient. Suitable compounds have the general formula:

[0021]

Embedded image

Here, X is phenyl or

[0023]

Embedded image

Where R is

[0025]

Embedded image

And n is an integer from 1 to 5;

[0027]

Embedded image

Y is t-butyl which can be hydroxylated or acetylated at one or more sites;
Phenyl; or

[0029]

Embedded image

Where W is

[0031]

Embedded image

And Z is a C ₁ to C ₅ linear or branched alkyl group.

5,5-dimethylpyrroline-N-oxide (DMP
O), or α- (4-pyridyl-1-oxide) -Nt-butyl nitrone (POBN); and other spin-trapping agents such as their spin-trapping derivatives may also be used.

Useful spin trapping compounds include the following compounds.

(PBN and its derivatives) A preferred spin trapping compound is α-phenyl-t-butyl nitrone (P
BN) and its derivatives. PBN has no measurable effect on normal or undamaged cells. Although PBN is currently the preferred compound, many of the compounds listed below are also useful. Especially 2-, 3- or 4-hydroxy PBN, and hydroxy derivatives such as mono-, di- and trihydroxy-t-butylnitrone; esters, especially 2-, 3- or 4-hydroxyphenyl-t- 2-, 3-, or 4- that release butyl nitrone
Phenylhydroxy nitrones, such as esters, ethyl derivatives, or acetoxy derivatives, such as carboxyphenyl-t-butyl nitrone; especially methyl derivatives, which are alkoxy derivatives that release 2- or 4-hydroxyphenyl-t-butyl nitrone. Acetamide derivatives such as acetyl derivatives that release 2- or 4-aminophenyl-t-butylnitrone; diphenylnitrones;
(DPN) and similar diphenylnitrone derivatives. The term "PBN" as used herein, unless otherwise indicated, refers to both phenyl-t-butylnitrone and its derivatives.

The general formula of PBN and its useful derivatives is:

[0037]

Embedded image

Wherein X is phenyl or

[0039]

Embedded image

Where R is

[0041]

Embedded image

And n is an integer from 1 to 5;

[0043]

Embedded image

Y is t-butyl which can be hydroxylated or acetylated at one or more sites;
Phenyl; or

[0045]

Embedded image

Where W is

[0047]

Embedded image

And Z is a C ₁ to C ₅ linear or branched alkyl group.

(Other spin trapping agents) 5,5-dimethylpyrroline-N-oxide (DMPO) or α- (4-pyridyl-
Other spin-trapping agents such as 1-oxide) -Nt-butyl nitrone (POBN), and spin-trapping derivatives thereof, may also be used. Derivatives are made using standard techniques such as, for example, replacement of a methyl group. The general formula for DMPO is:

[0050]

Embedded image

Here, A and B independently represent -CH ₃ , -C
H ₂ OH, -CH ₂ OW or

[0052]

Embedded image

Where n is an integer from 1 to 5, where W is

[0054]

Embedded image

Z is a C ₁ to C ₅ linear or branched alkyl group.

The general formula of POBN is:

[0057]

Embedded image

Here, Y is t-butyl which can be hydroxylated or acetylated at one or more sites;
Phenyl; or

[0059]

Embedded image

Where W is

[0061]

Embedded image

And S is -H,-(OR) n, where R is

[0063]

Embedded image

Where n is an integer from 1 to 4, or

[0065]

Embedded image

Wherein Z is a C ₁ to C ₅ linear or branched alkyl group.

[0067]

DETAILED DESCRIPTION OF THE INVENTION For the purpose of preventing or treating damage from ischemia, ATP may be administered prior to or during ischemia.
At a dosage effective to prevent or ameliorate predisposition to cell damage resulting from a reduction in the number of cells (as demonstrated by in vivo NMR) and from free radical generation after reperfusion Is administered. Based on animal studies, dosages for treating stroke injury can range from 10 to 300 mg /
kg range. The preferred method of administration for treating stroke is intravenous in humans. A preferred method of administration for the treatment of inflammation is direct delivery to the site of inflammation.

Examples of diseases that can be treated include stroke, meningitis,
Includes progressive neuronal loss from Parkinson's disease, senile dementia, and dysfunction resulting from drug abuse, exposure to hyperbaric or oxygen-rich environments, and hemorrhage to nerve tissue as a result of trauma .

For the treatment of aging, the composition is orally administered once or twice a day, 1 to 10 mg PBN / 70 kg
It is preferable to administer a dose corresponding to the human body weight. Animal studies have shown that administration of this compound for a period of two weeks reduces levels of oxidized brain enzymes to normal levels and restores memory to the same levels obtained in young control animals. Was. A marked decrease in oxidized protein and memory recovery was already observed 7 days after the start of treatment; from 1 to 3 days after discontinuation of treatment
Even after days, the level is comparable to the young control,
It decreased partially 7 days after discontinuation of treatment.

These compositions and methods may be used to treat age related dysfunction, pre-operative preparation and / or pre-anesthesia preparation, or administration of chemotherapeutic agents, and brain, cardiovascular, lymphatic system. It is useful for treating dysfunction or trauma, and potentially for treating certain viral dysfunctions characterized by oxidation of host proteins in cells infected with the virus.

As used herein, the term free radical scavenger, or spin trapping agent, is a molecule that forms a stable complex with free radicals. Free radical carbon traps are molecules in which free radicals are present on carbon atoms. As a result of this chemical bond formation, free radicals do not damage cells.

Treatment and Prevention of Ischemic Injury Following ischemia, there is a significant increase in free radical production (both oxygen and carbon-centered radicals). Early damage to brain cells is thought to be due to peroxide products, followed by secondary (carbon-centered) radical damage. Eventually, metabolic and synthetic pathways are damaged so that the cell dies. Spin-trapping derivatives of PBN and its functional equivalents that have crossed the blood-brain barrier block cell damage during or after ischemia by reducing or preventing ATP depletion, and are still present The oxygen radical spin trapping agent binds the free radical at the carbon center,
Shows the advantage of preventing further damage by modified enzymes or other components.

These compounds must also be very effective in the prophylactic treatment of chronic or cyclic cytotoxic conditions involving free radicals. Disease states of the CNS that are predicted to be treatable with such compounds include stroke, transient ischemic attacks, Alzheimer's gangrene, continuous cell loss in Parkinson's syndrome, meningitis, cardiac resuscitation- Includes induced brain injuries and injuries resulting from trauma, such as head and spinal cord injuries, especially with bleeding from surrounding tissue to nervous tissue. These compositions also include
Damage due to exposure to hyperbaric oxygen or oxygen-enriched environments, such as eye damage, particularly by placing extremely premature infants in hyperbaric oxygen inhalers, CNS damage induced by abuse of drugs and / or alcohol, and It is anticipated that it may be used to treat a variety of other dysfunctions, including exposure to ionizing radiation or damage from photo-oxidation.

(Effective Dosage and Administration of Spin Trapping Agents) α-Phenyl-t-butylnitrone (PBN) in a pharmaceutical solvent suitable for intravenous administration prevents CNS damage after ischemia or inflammation Or it is effective to recover. As used herein, the term ischemia is defined as a blockage of blood flow that causes damage to the central nervous system (CNS), including the spine and eye. PBN has many advantages, including being able to cross the blood-brain barrier and exhibiting no effect, such as having no measurable effect on normal or undamaged cells. The composition may also include other components such as tissue plasminogen activator, streptokinase, or other thrombolytic agents, other compounds that are oxygen radical scavengers, such as mannitol, or compounds that block peroxidase production, such as lazaroids. It may contain active ingredients.

The examples show the usefulness of this composition in preventing brain damage and death in animals following interruption of blood flow through the carotid arteries leading to the brain. For example, in gerbils, the dose of PBN is between 32 and 300 mg / kg body weight. The effective range of PBN in humans and other animals is about 1
It is between 0 and 300 mg / kg. The composition can be effectively administered before, during, after, and to prevent or reduce the degree of cell damage.

This trapping agent has little or no effect on normal cells, since trapping of endogenous free radicals occurs specifically only in cells that have been exposed to conditions under which free radical production occurs. This beneficial effect occurs only in damaged cells. Furthermore, since this beneficial effect does not require the presence of specific receptors or specific enzymes, the spin trapping agent provides a significant improvement in the treatment of ischemia-induced cell dysfunction and gangrene.

The PBN is preferably administered systemically,
Intravenous or oral administration is most preferred because it is the quickest and most effective way of directing the active compound to the site of free radical generation. However, in the example
Other methods of administration may be used, such as PBN administered intraperitoneally or intranasally into the pulmonary vein.
Alternatively, PBN is injected intramuscularly or subcutaneously, ointment,
Alternatively, it may be administered locally via a sustained release implant.

Pharmaceutical carriers suitable for intravenous administration include:
Physiological saline at physiological pH or phosphate buffered saline. In a preferred method, PBN is delivered to the patient intravenously at a dose near 10 mg / kg / hr, with a range of 1 to 3 mg / kg / hr being most preferred. The dosage for a particular disease state and / or species is determined by measurement, as described in detail below. Generally, an effective dose will reduce enzyme activity loss by 25%, reduce cell death by 25%, and / or maintain normal function by 25% or more,
The amount of PBN.

The present invention provides a method for determining an effective dose of PBN for the treatment of injury caused by ischemia, including but not limited to the following: It will be better understood with reference to examples, which have shown a recovery.

[0080]

EXAMPLES A Gerbil Stroke Model A validated animal model for determining the effects of compounds on injury caused by stroke is described in Chandler et al . , J. Pharm. Meth.
ods 14, 137-146 (1985). In summary, Mongolian gerbils are pentobarbital (40mg
/ kg). Make a ventral midline incision in the neck. The common carotid artery is exposed and separated from the vagus-sympathetic trunk.
Wrap an unwaxed dental floss (Johnson and Johnson) looped around each carotid artery. Each end of the floss is a double lumen catheter (Dural
Plastics and Engineering, Dural, NSW, Australia)
Through with one lumen. The catheter and dental floss penetrate the muscles of the back and are stimulated on the back of the neck. The catheter is secured at a location just above the carotid artery using a cyanoacrylate adhesive at the drain.
During the daily lavage and exploratory activity tests, the length of the dental floss is marked to ensure that the carotid artery is not occluded. The ventral midline incision is closed with a 9 mm wound clip. 48 hours after placement, the looped dental floss is slowly pulled until the carotid artery is occluded, causing ischemia. Occlusion of the carotid artery is accompanied by loss of consciousness, altered pituitary and respiratory patterns. In previous studies using direct observation,
Under these conditions, complete obstruction of blood flow occurred. Blockage
Dental floss was removed for 10 minutes and then completely reperfused. After reperfusion, the catheter was trimmed flush with the surface of the neck.

The animals were male gerbils (50-60 grams) purchased from Tumblebrook Farm. West Brookfield. MA and kept in groups of three at least one week prior to surgery. Following surgery, they were housed in one to avoid accidental ischemia induction by cagemates. Food and water were fed ad libitum in a home gauge. All gerbils were placed on a 12 hour light / dark cycle.

Example 1 Measurement of Progression of Peroxidative Damage in Brain Tissue Measurement of Tissue Damage by Generation of Free Radicals Tissue damage is caused by generation of free radicals, increase in protein carbonyl concentration, and enzyme activity of glutamine synthetase Can be measured as a function of the decrease in

Free radicals are found in the gerbil brain with ischemia / reperfusion injury. There are several effective methods for measuring free radical generation, including spin trapping and salicylate hydroxylation. Salicylate hydroxylation can be used to monitor hydroxyl free radicals in vivo. Because of the hydroxylation products (2,5- and 2,3-dihydroxybenzoic acid, DHBA)
Is present in the gerbil brain pretreated with salicylate. Cao et al., Neuroscience Let
As shown at t , 88, 233-238 (1988), the amount of DHBA is closely related to the degree of brain damage observed in this model. The oxidative damage of the protein that occurs in the gerbil brain can be assessed by increasing the carbonyl groups of the protein. This increase in carbonyl groups occurs when reperfusing the brain of ischemic damaged gerbils. In addition, necrosis of whole tissues is indicated by reduced enzymatic activity of glutamine synthase (GS) in the brain of ischemic injured gerbils. This decrease progresses as the reperfusion time increases.
35% reduction. The enzymatic activity of GS is sensitive to metal-catalyzed oxidative damage. Thus, this loss of enzyme activity supports the notion that metal-catalyzed oxidative damage occurs during the reperfusion phase of the gerbil with ischemic damage. GS enzymatically converts glutamate to glutamine, and thus reduced activity can lead to the accumulation of glutamate, an excitotoxic amino acid.

Using ischemia / reperfusion injured gerbil brain, the occurrence of oxidative damage was confirmed with the following evidence: 1) associated with hydroxyl free radical efflux in the damaged brain. Increased levels of salicylate hydroxylation products; 2) increased levels of protein oxidation and loss of glutamine synthetase activity in the injured brain; 3) formed from spin traps in the injured brain. Free radicals; 4) spin-trap mediated prevention of ischemia / reperfusion injury of the brain; and 5) increased brain peroxidative capacity during the reperfusion phase following an ischemic attack.

Spin Trapping Using Electrochemical Detection
Oxygen free radical flow in vivo by HPLC and HPLC
Spin trapping and HPLC by electrochemical detection (LCED) for the determination of the hydroxylation products of salicylate partially formed by the addition of hydroxyl free radicals and the hydroxyl free radical adducts to DNA and RNA Using. The LCED method, which is 10 ³ to 10 ⁴ times more sensitive than the optical method, stands out when attempting to identify oxygen free radical efflux in vivo, reaching only 10 ^-9 M under high oxidative stress conditions There are advantages. It was confirmed in biochemical systems that OH radicals could be trapped by salicylate and quantified using LCED.

Electron spin resonance of the lipid extract of the brain was measured as follows. For gerbils, one hour before treatment,
A spin trap stored in a cold place (shielded) dissolved in physiological saline is administered by IP. The gerbils are subjected to ischemia / reperfusion treatment or sham-operated as a control and transferred to an animal holder for a period of time without deoccluding for a period of time.
Kill a gerbil by decapitation. The brain is removed and placed on a chilled table for no more than 2 minutes. The brain cortex is removed and stored refrigerated at 8 ° C. for approximately one week until extraction. The cortex is homogenized in 0.5% NaCl (0.5 gm / ml) solution at ice temperature,
Next, the homogenate was mixed with chloroform-methanol 2: 1.
Extract with the mixture (V / V) and make a final dilution to 20 times the volume of the tissue sample. The crude extract is washed thoroughly with 0.5% NaCl (5: 1, V / V) and the mixture is kept at 4 ° C. for 2 hours and separated into two layers. The organic layer was collected, or obtain the concentrations required by bubbling with N _2, or dried by solvent evaporation of the organic layer and the residue redissolved in chloroform. Finally, transferring the sample to a Pasteur pipette (capillary end is sealed) is bubbled for 5 min N _2. The pipette is then transferred to the sample compartment of an IBM Bruker ESP300 EPR spectrometer and scanned for the presence of spin adducts. The normal setting of the spectrometer is as follows: microwave power 19.8
mW; modulation intensity 0.975G; time constant 1310.72 ms; scan range
100G; and scan time 6 minutes. All spectra are recorded at room temperature. The coupling constant is obtained directly from the instrument,
Since the apparatus is controlled by an on-line computer, a simulated spectrum of a mixture of spin-trapped free radicals can be compared to the apparent spectrum obtained to obtain predictions about the trapped free radicals. An extensive database of spin coupling constants obtained in different solvents using different systems is used to compare the results.

The spin trap reacts with free radicals to form an adduct. Electron spin resonance (ESR) spectra provide a mechanism for estimating the species of trapped free radicals. The method used for in vivo spin trapping is described in Lai et al . , Arch. Bioch.
em. Biophys. 244, 156-160 (1986), developed by Dr. PB McCay and Dr. Ed Janzen. Bolli and McC show that free radicals were spin-trapped in ischemia / reperfusion injury heart in rats
ay, J. Clin. Invest. 82, 476-485 (1988).

The production of free radicals during the period of injury that induces ischemia / reperfusion in the gerbil brain was tested by spin trapping. Prior to experimental treatment, spin-trap PBN was added to both treated and control animals.
(Α-phenyl-t-butylnitrone) was administered. Data show that large signals of free radicals were obtained from ischemic / reperfused gerbil brain extracts, in contrast to very small signals from sham-operated control animals. Surprisingly, these radicals are not what is expected when a spin trap reacts with a free radical to produce a spin adduct. 6
Multiplet spectra are expected from the free radical spin adduct of PBN. The spectrum of the hex line is due to the nuclear magnetic moment of the proton spin 1/2 and the free electrons interacting with spin 1 from the nitrogen of the N-oxide group. In contrast, the spectrum obtained is a triplet only, and thus this spectrum is due to free electrons interacting only with the nitrogen of the N-oxide group. This means that nearby protons have moved to a distance that does not affect free electrons.

In response to this result, PBN first traps free radicals, presumably lipid carbon free radicals, and then oxidizes the spin adducts into nitrones that trap other free radicals, and reduces the observed spectrum. The explanation is most likely that a nitroxide with the resulting properties is obtained. Alternatively, it can be explained that PBN is metabolized to methyl nitrosopropane (MNP) and the t-butyl radical is trapped to produce t-butyl nitroxide.

The other two spin traps, DMPO (5,5-dimethylpyrroline-N-oxide) and POBN (α-pyridyl-1-oxide-Nt-butylnitrone), both differ in chemical structure from PBN, Provided the same nitroxide spectrum (very similar triplet with slightly different nitrogen coupling constants and different intensities) obtained with PBN in ischemia / reperfusion treated gerbils . When the chloroform / methanol extract of the brain of the animal pretreated with PBN is left at room temperature for 24 hours, the signals of the three lines disappear. However, PB
When N is added and incubated for 24 hours, a trapped radical signal is observed in the ischemia / reperfusion extract, but no signal is seen in the sham-operated control. Computer simulations of the evolution of the signal indicate the presence of three radicals. One has parameters equivalent to the triplet nitroxide previously observed, and the other two are spin trapped carbon free radicals, possibly lipid radicals. These observations indicate that oxidation is initiated in the ischemic brain but not in sham-operated controls, indicating that these events are at least partially carried over to lipid extracts.

Ischemic / reperfusion induced increased brain peracid
The homogenates demonstration brain of ability, but is not a natural peroxide in the ice temperature,
Raising the temperature to 37 ° C. results in spontaneous peroxidation, in contrast to most other tissue homogenates. The rate of peroxidation varies from brain section to brain section and is strongly correlated to the total iron content of that brain section. Gerbil homogenates peroxidize faster than rat brain. The susceptibility of brain homogenates to peroxidation following ischemia / reperfusion injury and the effect of reperfusion time were measured. Data was obtained from 176 gerbils. At each time point, sham-operated animals were used as controls. The data show that after 10 minutes of ischemia, there is a reduction in the rate of peroxidation if no reperfusion occurs. Upon reperfusion after ischemia, the rate of peroxidation is time dependent,
It increases significantly over sham-operated control animals. The increase is fastest in the first 60 minutes, then slows down,
It peaks on day 7 of ischemia and then decreases, but its value is still higher on day 14 than in controls.

The gerbil brains pretreated with pentobarbital, then ischemic for 15 minutes, and then reperfused for 15 minutes, have no increase in spontaneous peroxide levels over sham-operated controls. Thus, pentobarbital not only protects gerbils from damage caused by ischemia / reperfusion injury, but also has no increased peroxidation in brain homogenates from pentobarbital-treated animals compared to untreated animals. . In animals pre-treated with 15 min ischemia / 15 min reperfusion injury, spontaneous peroxidation of brain homogenate is approximately 15% higher than sham-operated controls and directly correlates with increased brain homogenate peroxidation Is shown.

Example 2: Demonstration of Protective Effect of Spin Trap in Ischemia / Reperfusion Induced Gerbil Brains Spin Trap PBN significantly modifies gerbils from brain damage caused by ischemia / reperfusion injury Helps protect and even prevent deaths often associated with brain damage. Measurement data proving these descriptions is shown in FIGS.

FIG. 1 is a graph of a dose-effect curve for the beneficial effect of PBN on transient ischemia-induced hyperactivity, plotting migratory activity versus dose in mg / kg of PBN. . Young adult gerbils received a single dose of PBN one hour before bilateral carotid occlusion for 5 minutes. twenty four
After time, changes in voluntary activity were measured. The control value (CON) represents the level of exploratory activity of a group of gerbils without normal knowledge. Physiological saline (SA
L) was measured in gerbils injected with saline one hour before ischemia. Data were expressed as the mean (± SE) of 6 animals per treatment group.

[0095]

[Table 1]

Table 1 shows a group of animals that were administered IP at a dose of 300 mg / kg IP of PBN dissolved in saline 60 minutes before ischemia for 15 minutes and then reperfused for 24 hours. This group of animals was protected from death and indicates the number of animals that survived beyond the estimated seven days of death. 50% of young gerbils and 100% of old gerbils in the control group of animals that received saline died by day 7.

Gerbils that have been permanently injured by cerebral ischemic injury first show a lethargic response and then at 4 hours after ischemia a characteristic increase in spontaneous locomotor activity over the control group. showed that. The hyperactivity induced by this injury
Lasts several days. Agents that protect animals from ischemia / reperfusion injury prevent this characteristic appearance of spontaneous activity. PBN
Was effective at various doses in protecting against hyperactivity-induced ischemia / reperfusion, as shown in Table 1 and FIG. It can be seen that saline-treated animals that have been subjected to a 5 minute ischemic injury have significantly greater hyperactivity 24 hours after ischemia than sham-operated controls.
However, animals receiving 32 or 100 mg / kg of PBN one hour prior to ischemia did not have elevated hyperactivity.

[0098] 300 mg / kg PBN reduced activity compared to controls. 24-hour reperfusion of 2 min ischemic, 5 min ischemic and 15 min ischemic injured animals receiving either saline or 300 mg / kg PBN and sham-operated controls for 24 h Data comparing activity over time,
As shown in FIGS.

FIG. 2 is a graph of the effect of PBN on the level of activity during a continuous 10 minute interval in the test shown in FIG. Data are presented as the mean (± SE) of 6 animals per treatment group;

[0100]

[Outside 1]

FIG. 3 is a graph of the effect of PBN on post-ischemic activity activity of aged gerbils (at least 20 months of age) tested 24 hours after ischemia at 2-minute intervals, as described in FIG. It is.

[0102]

[Outside 2]

Is as follows.

FIG. 4 shows 300 mg PBN / kg PB against changes in voluntary behavioral activity of gerbils induced by 15 minutes of ischemia.
6 is a graph of the effect of N. Activity activity is plotted against time (minutes). The physiological saline treatment group (●-●)
10 control gerbils (◆-
◆) represents 5 of them.

[0105]

[Outside 3]

All data were expressed as mean values (± SE). If SE is not indicated, SE is less than the size of the symbol.

The above results clearly indicate that high administration of PBN reduces ischemia-induced hyperactivity to control levels or below.

To obtain parameters for the acute toxicity of PBN, the total cumulative activity of normal gerbils was examined at a wide range of concentrations. The data is shown in FIG.

FIG. 5 shows the result of comparison of the accumulated behavior activity count values for successive 10-minute period intervals. Administered saline

[0110]

[Outside 4]

Is as follows.

As can be seen, there was a notable change in activity only at concentrations of PBN of 1000 mg / kg and higher. 2000 mg / kg PBN was not lethal. The only recorded effect of high PBN is a lethargic response that disappears after 24 hours.

The effect of PBN administration on the survival rate of animals given a lethal stroke (15 minutes of ischemia) is shown in FIG. FIG.
FIG. 4 is a bar graph comparing the survival rates of gerbils treated with saline or 300 mg PBN / kg after lethal occlusion of the carotid artery. As can be seen, 50% of the animals that received 15 minutes of ischemia died. Ischemic pretreatment with PBN survived 100% of the animals. Ischemia / reperfusion induced oxidative damage to brain proteins.

The gerbil brains that underwent various treatments were
Protein carbonyl groups, which are indicators of oxidative damage to proteins, and glutamine synthetase activity were analyzed. The results are shown in FIGS. 7 and 8 and FIG.
Was compared.

FIG. 7 shows the oxidative damage to proteins as measured by carbonyl content in reperfusion at the indicated times as a percentage of the carbonyl content of the control gerbils given 10 minutes of ischemia.

FIG. 7 shows gerbils undergoing ischemia, reperfusion for 15 minutes after ischemia, reperfusion for 60 minutes after ischemia, and pre-ischemic administration of 300 mg PBN / kg for 60 minutes after ischemia. 5 is a graph comparing% of carbonyl protein to control.

300 mg PBN / kg was administered 1 hour before the onset of ischemia. In brains that had been subjected to ischemia for 10 minutes and were not reperfused, carbonyl protein content increased above control levels. Reperfusion for 15 minutes did not cause the carbonyl protein to exceed that of ischemia alone.
After 60 minutes of reperfusion, there was a significant increase in carbonyl protein. Pretreatment of PBN prevents this rise.

FIG. 8 shows the oxidative damage to glutamine synthetase activity at reperfusion for the indicated times as a percentage of gerbils of controls given 10 minutes of ischemia.

FIG. 8 shows gerbils undergoing ischemia, reperfusion for 15 minutes after ischemia, reperfusion for 60 minutes after ischemia, and pre-ischemia at 300 mg PBN / kg and reperfusion for 60 minutes after ischemia. 5 is a graph comparing the percentage of glutamine synthase of the present invention to the control.

[0120] 300 mg PBN / kg was administered one hour before the onset of ischemia. Glutamine synthetase (GS) activity was slightly lower than control without reperfusion after ischemia. As the reperfusion time increased, the GS activity decreased dramatically, leaving only 65% of the activity after 60 minutes of reperfusion. Animal PBN
Pretreatment prevented this decrease in GS activity. Since this enzyme is considered a glial cell marker,
It shows post-ischemic damage that also occurs in glial cells.

FIG. 9 is a graph correlating carbonyl / ml protein versus glutamine synthetase specific activity. Control (□); 10 min ischemia, no reperfusion (□) 10 min ischemia, 15 min reperfusion (□), 10 min ischemia,
60 min reperfusion (□) and 10 min ischemia, 60 min reperfusion and PBN (■).

FIG. 9 shows that there is a good correlation between loss of GS activity and accumulation of carbonyl protein when comparing all samples analyzed.

Ischemia / Reperfusion-Induced Gene Expression In the gerbil brain cortex after ischemia / reperfusion, c-fo
The mRNA levels of s and c-jun were examined. 60 minutes after 10 minutes of ischemia
Investigations at minute reperfusion show that both c-fos and c-jun mRNA levels rise within 60 minutes after injury. Low c-fos m in control and surgery animals
RNA levels increased significantly. Pentobarbital pretreatment, which protects animals from ischemia / reperfusion injury, prevents the induced expression of the c-fos gene, whereas without pretreatment, c-fos mRNA is greatly increased. PBN pretreatment suppresses evoked levels of c-fos in ischemia / reperfusion injury.

As shown in Table 2, older gerbils are more sensitive to cerebral ischemia / reperfusion injury than younger animals.
Data are shown as survivors / total tests.

[0125]

[Table 2]

Example 3 Efficacy of Spin Trapping Agents Other than PBN The effectiveness of two other spin traps, DMPO and POBN, in preventing the acute effects of ischemic damage on the brain was tested.
This result indicates that DMPO is even less effective than POBN, which is less effective than PBN, but both DMPO and POBN exhibit some protection.

Treatment of Aging Spin-trapping agents have also been found to be useful in the prevention or treatment of symptoms associated with aging, trauma, drug administration and surgery, especially of the brain. For administration to patients,
In combination with pharmaceutical excipients, preferably orally, these spin-trapping compounds reduce aging-related symptoms, such as increased oxidized protein levels, decreased enzyme activity, and spatial and short-term memory. It is useful for preventing or recovering. To date, no non-toxic treatment is effective against aging. Efficacy is shown in animals after only 7 days of administration. Efficacy lasts for at least one week after administration. Values return to pre-treatment levels after 2 weeks.

In one embodiment, a spin trapping agent is administered to a patient to ameliorate damage that occurs as a function of aging. As a preliminary result, in the brain,
It is shown that there is a substantial increase in protein oxidation and accumulation of oxidants. Further, the occurrence of senile plaques is routinely observed in elderly patients.

Other abnormalities include traumas such as head bruises, drug treatments such as administration of anesthetics or drug abuse, or abnormalities resulting from some type of viral infection.

It has been determined that oxidized protein levels in the brain are inversely proportional to the ability of short-term memory function and directly proportional to the risk associated with stroke-induced damage and altered behavior. Increased cellular oxidation can result from one or more of the following: (a) oxidative damage of cellular proteins that can result in altered regulation of ion channels; (b) the rate and rate of signal translation and membrane depolarization. Changes in efficiency; (c) significant changes in energy output that could impair selective function; (d) changes in RNA transcription due to oxidative damage of DNA; (e) RNA translation is either RNA or regulatory macromolecules. May be affected by oxidation; or (f) the rate of protein degradation may be altered.

Any of these changes can negatively impact the consolidation and retrieval of information as it is acquired, even if it interferes with one of the learning and memory processes. Oxidation of cells in certain brain regions can result in learning deficits, and oxidation of other regions can result in defective output. In view of the many destructive neurodegenerative diseases, including Alzheimer's disease, the treatment of the present invention can provide a great deal of help to patients with these dysfunctions.

Examples of other dysfunctions that can be treated with the compositions of the present invention include diabetic peripheral neuropathy, exercise-induced muscle damage and pain, and enhanced cellular responses to hormonal signals. .

Treatment of Neurodegenerative Dysfunction Some neurodegenerative conditions are best treated with compounds that interfere with the oxidation of proteins. Alzheimer's disease is associated with abnormal accumulation of oxidized proteins or production of abnormal proteins in pathologically affected areas. In addition, the promotion of aging-associated protein oxidation occurs in all cells of elderly individuals. PBN and its derivatives have been shown to be useful in reducing protein oxidation and increasing the activity of key enzymes in the brain of aged animals. Since this is a fundamental change in oxidation state, PBN
And other related compounds are useful in providing early stage individuals with Alzheimer's disease, or perhaps even end stage individuals, with prolonged periods of time. In addition, many infarct dementias can also be treated with these compounds. This is because these dementias are also caused by ischemia-reperfusion oxide.

Senile dementia has not been directly evaluated as the pathogenesis of ischemia reperfusion or protein oxidation, but senile dementia can also be treated with these compounds. This is based on the hypothesis that aging is associated with increased production of oxidized proteins. In the unique state of aging, where aging is accelerated, Stadtman et al., NIH researchers,
In older patients, even young adults showed a marked increase in basal levels of oxidized proteins. This is described in Oliver et al., J. Biol. Chem. 262, 5488-5491 (1987).
And Starke-Reed and Oliver, Arch. Biochem. Biop
hys. 275, 559-567 (1989). Although this is a rare condition, it can also be treated by the compositions of the present invention.

Another condition that may be related to oxidative damage resulting from microcirculation difficulties is diabetic peripheral nerve disease and vascular deformity. These are disastrous conditions that ultimately require amputation to help the patient. These compounds do not appear to improve vascular flow, but result in oxidative and peripheral nerve damage, and in addition, vascular flow, which results in skeletal muscle damage, a condition often referred to as exercise-induced or intermittent claudication. It appears to reduce the impact of transient deformation. If retinopathy associated with diabetes is a problem of ischemia reperfusion of the microcirculation, spin trapping compounds are useful in treating retinal damage that occurs very frequently in diabetics.

Preoperative Preparations Cell status and viability in a hypoxic or anoxic environment, as opposed to peroxidation, depends on the ability of cells to compartmentalize metals and to process oxygen in useful ways. So
These compounds are expected to be useful as preoperative preparative medical treatments that reduce carbonyl load and improve patient enzymatic status prior to elective surgery. In addition, these compounds help somatic cells achieve higher levels of enzyme function, shorter recovery times, and further reduce the potential for any intraoperative difficulties associated with changes in microcirculation.

Treatment of Viral Infections and Inflammatory Disorders Retroviruses selectively infect certain types of cells, such as lymphocytes. One example of a retrovirus that has been the subject of much research is the human immunodeficiency virus (HIV), which causes the immunodeficiency syndrome (AIDS). There have been many attempts to find a cure, but no cure for infection has yet been found. Since lymphocyte activation is involved in the oxidation of proteins, and the activation of lymphocytes is required prior to the release of newly formed virus, administration of these compounds may lead to infection by the virus and lymphocyte activation. Expected to inhibit replication. Activation of T-4 lymphocytes
A cascade of biochemical intracellular changes, one of which is involved in the production of oxidized proteins. If a PBN-related compound can block protein oxidation in an anomalous manner, then PBN or other spin-trapping compounds may replicate the virus and / or transfer the virus from host cells (T-4 lymphocytes). It can interfere with the process of dispersal and act as a virus stasis agent by preventing T-4 lymphocytes from releasing newly formed virus. This is similar to the use of isoniazid (INH) in the treatment of tuberculosis. INH reduces tuberculosis infectivity and dispersal at low doses, arrests tuberculosis, thereby effectively preventing damage to the patient's lungs.

It is noted that the effects of the spin trapping compound occur in animals with basal levels of carbonyl formation that may be required for post-translational processes. Older animals have a significantly increased level of carbonyl proteins associated with decreased enzyme function compared to younger control animals. When exactly the same dose is given to young control animals, there is no significant change in the enzyme activity and no significant change in the protein carbonyl. Therefore, PB
N and related spin trapping compounds do not appear to interfere with the basic processes required for normal cell function.

In conclusion, many clinical conditions appear to have cellular protein oxidation and enzymatic damage as underlying causes. Spin trapping compounds are effective in animal models to reduce protein oxidation and improve enzyme function. This occurs in specimens that have modified and protected abnormally oxidized proteins so that normal post-translational oxidation occurs. This suggests that PBN does not interfere with the oxidation of normal, necessary proteins after synthesis.

The effective dose of the spin trapping agent and
Methods of Administration Examples of doses of PBN are 0.1 to 10 mg / kg of animal body weight. Effective amount of human PBN is about 1 to 10m / 70kg
It is considered between g. In a toxicity test, this compound showed LD
_Fifty was shown to be indeterminately low toxicity and completely harmless.

In a preferred application, PBN or other spin wrapping agents are administered to patients who often have memory loss or other symptoms associated with aging. Optimal results are generally achieved with oral administration daily or twice daily.
Observed after a week. This composition can be used before surgery, during surgery,
Alternatively, it can be effectively administered immediately after surgery and can prevent or reduce the spread of cell damage from either trauma or anesthesia.

The trap has little or no effect on normal cells, since trapping of endogenous free radicals is specific only to cells that have been exposed to conditions that lead to the formation of free radicals. The beneficial effects only occur on the damaged cells and do not require the presence of specific receptors, specific enzymes, and / or specific cell types.

PBN or other spin trapping agents are preferably administered systemically, most preferably orally. This is because this is the fastest and most effective way of delivering the active compound to the site of free radical generation. The PBN may be administered at once, or may be divided into small doses and administered at various time intervals. Other modes of administration, including subcutaneous, intravenous, and intraperitoneal administration, can also be used. The concentration of the active compound in the drug composition will depend on the rate of drug adsorption, inactivation and elimination, as well as other factors known to those skilled in the art. Further effective doses also
It is determined based on the amount required to prevent or restore the cell's predisposition to damage due to ATP reduction (as indicated by in vivo NMR) and damage due to free radical generation. It is also noted that dosage values will vary with the condition of the patient being treated. It will also be appreciated that for any given individual, the particular dosage regimen will be adjusted over time according to the needs of the patient and the professional judgment to administer or direct the administration of the composition. Accordingly, it is also understood that the concentration ranges set forth herein are exemplary only, and are not intended to limit the scope or practice of the claimed compositions.

A preferred mode of administration of the active compounds is in a form for oral delivery. Oral compositions generally include an inert diluent or an edible carrier. Preferred pharmaceutical carriers for intravenous administration are saline or phosphate buffered saline at physiological pH. PBN is p
Since H decomposes below about 3 to 4, PBN is preferably administered at pH 4 or higher, or in combination with food and buffers, or enteric-coated. For oral delivery, PBN alone or in combination with immobilizing matrix materials such as starch or low-absorbable salts such as immodium is made into a solution or suspension, enclosed in capsules, compressed into tablets, and microcapsules. Can be incorporated into liposomes. A pharmaceutically suitable binding agent may be included as part of the composition. Tablets or capsules, for example, may contain a compound of any component or similar nature following: a binder such as microcrystalline cellulose, Torogakangamu or gelatin; starch and an excipient such as lactose; alginic acid, Primogel ^R,
Or a disintegrant such as corn starch; a lubricant such as magnesium stearate or Sterotes; a lubricant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

The present invention is directed to treating aging related syndromes,
Alternatively, it can be further understood by reference to the Examples which show how to determine the efficacy of PBN administration for prevention and / or recovery. However, it is not limited to this embodiment.

Example 4 Determination of Enzyme Levels in the Brain of PBN-treated Gerbils at Old and Young Ages, Duration of Ischemia and Changes in Both Spontaneous Behavior or Changes in Oxidized Protein Levels in the Brain Correlations have previously been shown in gerbils. Many other psychiatric and neurological conditions have been proposed to be the result of oxidation. Among these conditions, cellular senescence is associated with the accumulation of oxygen radicals and proteins. Comparison of oxidized protein levels, glutamine synthetase activity, brain protease activity, and radial arm maze test in young adult gerbils and ex-reproductive gerbils, determined by protein carbonyl assay, Age and brain oxidized protein levels were shown to be directly proportional. Increased protein carbonyl levels were associated with decreased glutamine synthetase activity and decreased alkaline protease activity. In contrast, as compared to young adult gerbils, the acidic protease activity of gerbils that had escaped the reproductive phase did not change. Consistent with an age-related increase in protein oxidation and enzyme inactivation, compared to young adult gerbils, gerbils that escaped the reproductive period made significantly more mistakes in short-term memory tests. These studies have shown that loss of function as a result of aging may be associated with increased protein oxidation and decreased enzyme activity in the brain.

This system was used to demonstrate the effectiveness of PBN in restoring old animals to restore enzyme levels and short-term memory in young brains. This result is shown in FIG.
It is shown in FIG.

FIG. 10 shows the relationship between the alkaline protease activity from the cortex of gerbils (3 to 4 months old young gerbils relative to the cortex), young gerbils (3 to 4 months old) and old gerbils (former breeders). Of 12
To 15 months of age), aged gerbils injected twice daily with 0.1 ml saline / kg body weight, and old age receiving 10 mg PBN / kg body weight twice daily in saline for 2 weeks 3 shows alkaline protease activity of gerbils. Protease activity was measured using oxidized proteins extracted from the cerebral cortex of young gerbils.

FIGS. 11A and 11B show that the carbonyl activity (pmol / mg protein) (A) and the glutamine synthetase activity (B) of the protein were measured at 32 mg PBN twice a day.
FIG. 3 is a graph comparing the cerebral cortex (neocortex) of young adult and old gerbils administered / kg at day 1, 3, 7, 7 or 14 days after administration. At the end of each indicated day, the animals were decapitated and the cerebral cortex was removed and snap frozen in liquid nitrogen. The carbonyl content of the protein was measured using the DNPH method. This result indicates that reduced oxidative damage to proteins in the gerbil cortex, and loss of enzyme activity, was caused by 32 mg P
It shows what happens as a result of BN / kg (ip). Each histogram is the average of three samples.

FIGS. 12A, 12B and 12C show two times a day.
FIG. 6 is a graph comparing the time course of protein carbonyl (pmol / mg protein) (A), glutamine synthetase (B), and protease activity (% of control) (C) after stopping administration of 10 mg PBN / kg body weight. is there. FIG. 12A shows the levels of carbonyl in soluble protein obtained from gerbils treated for 14 days, 1, 3, 7 and 14 days post-dose. FIG. 12B shows the relationship between the time of decrease in cortical glutamine synthetase (GS) activity after stopping the injection of PBN twice a day. FIG. 12C shows two times a day.
FIG. 4 shows the relationship with the time of decrease in alkaline protease activity after stopping injection of PBN. Each histogram was represented by the average value ± standard error (SE) of three samples at the indicated times. * And the dashed line indicate the value of the old untreated control for each measurement.

FIGS. 13A, 13B and 13C show twice a day,
FIG. 7 is a graph comparing the time course of protein carbonyl (pmo1 / mg protein) (A), glutamine synthetase (B), and protease activity (% of control) (C) after stopping administration of 32 mg PBN / kg body weight. is there.
FIG. 13A shows the levels of carbonyl in soluble protein obtained from gerbils treated for 14 days, 1, 3, 7 and 14 days after administration.
FIG. 13B shows the relationship between the time of decrease in cortical glutamine synthetase (GS) activity after stopping the injection of PBN twice a day. FIG. 13C shows the relationship between the time of decrease in alkaline protease activity after stopping the injection of PBN twice a day. Each histogram was represented by the average value ± standard error (SE) of three samples at the indicated times. * And the dashed line indicate the value of the old untreated control for each measurement.

FIG. 14 is a graph of an eight-arm radial maze experiment of young or old gerbils treated with PBN or saline. Gerbils were placed in the central chamber of a maze with barriers that limited exploration. After removing the barrier, the number of arms re-entered and the time elapsed to enter all eight arms were recorded. Each histogram represents the mean ± SE of 18 animals. Each animal receives PBN twice a day,
Doses were administered for 7 days (either 10 or 32 mg PBN / kg body weight) and tested at the end of day 7.

Young gerbils are from Tumblebrook Farms,
Obtained from West Brookfield, Mass. And weighed 5
At 0-60 grams, the age was 3 to 4 months. Animals were decapitated and sacrificed, and brains were removed for analysis.

FIG. 10A shows a young (3 to 4 months) gerbil, an old (12 to 15 months after reproductive period) gerbil, and 0.1 ml of physiological saline twice a day (b .
id) aged gerbil, and 10 mg PBN / body weight
1 is a graph showing the percentage of aged gerbils alkaline protease administered kg for 14 days in bid.

The results show that PBN is effective in restoring alkaline protease levels in old animals to levels present in young animals. A of FIG.
FIG. 2 is a graph plotting changes in protein oxidation in the brain of young and old gerbils versus nmol protein mg / mg protein versus days of PBN treatment.
Gerbils were injected twice daily with 32 mg / kg PBN for 14 days. Animals were sacrificed on days 1, 3, 7, and 14 and protein carbonyl levels were measured.

As can be seen, there is no change in oxidized protein levels in young gerbils treated with PBN up to 14 days. This indicates that this level of oxidized protein is a result of normal and necessary post-translational effects, such as, for example, after protein synthesis, when the protein is activated by modifications involving carbonyl oxidation. It seems that it is likely. In contrast to carbonyl levels in young animals, control older animals (15 months of age) had a marked increase in carbonyl content. This increase in carbonyl content corresponds to PBN treatment. Multi-day treatment of PBN reduced protein carbonyl levels to levels found in young animals.

The decrease in protein carbonyl levels (oxidative protein load in the nervous system) is up to the level in the brain of young gerbils. Both levels in the brain of young gerbils and in the brains of old gerbils cannot be reduced beyond this level. This observation indicates that there is a required level of oxidation that occurs in cells of normal animals, which is required for the cells to have "normal function", and that the control aged animals (15 months of age) have a significant increase in carbonyl content. There is a hypothesis that there is. This increase in carbonyl content corresponds to PBN treatment. The ability of PBN to reduce the cellular carbonyl load of a cell is an oxidative process that occurs at normal or expected rates, and if this process is interrupted, a mechanism by which this oxidized protein can be eliminated is a mechanism within the brain cells. It is shown that there is.

FIG. 11B shows a comparison of glutamine synthetase (GS) levels in young and old animals and the evaluation of the effect of daily administration of PBN on the specific activity of the enzyme. This particular enzyme marker was selected because it is a protein that is highly sensitive to oxidation and that is a metal-bound metal protein that can participate in the generation of free radicals when the metal dissociates from its binding site. Was. Glutamine synthetase activity
n and co-workers (Oliver et al . , Proc. Natl. Acad. Sc.
i. USA 87, 5144-5147 (July 1990)) as a marker enzyme for changes after protein oxidation.

As shown in FIG. 11B, glutamine synthetase levels were reduced in young adult gerbils (2.
Lower in older gerbils (1.2) than in 1). This is consistent with previous studies where increased carbonyl protein levels (oxidized proteins in cells) were associated with decreased glutamine synthetase activity. In particular, if the glutamine synthetase enzyme was purified and the carbonyl content of the enzyme was assessed, there would be a marked increase in oxidized protein levels with the presence of a decrease in glutamine synthetase activity. As further shown in FIG. 11B, repeated administration of PBN to young gerbils had no effect on glutamine synthetase activity, providing further evidence that this carbonyl level is associated with normal function. Provided, long-term administration of PBN does not affect carbonyl levels and marker enzyme activity. In contrast to young gerbils, older gerbils injected daily with PBN paralleled the decrease in protein carbonyl content,
Shows a time-related increase in glutamine synthetase activity. This indicates that reduced cellular oxidized protein load is associated with the restoration of enzyme activity to normal levels found in young adult gerbils.

It is important to note that there is no increase in enzyme activity beyond that seen in young adult gerbils. Thus, PBN treatment reverses the effects of aging on enzyme activity, but does not result in activation of enzyme activity above normal.

Example 5: Determination of the residual effect of PBN on the reduction of enzyme levels in the brain As shown in FIGS. 12A, B and C, FIG.
Administration of 10 mg PBN / kg body weight to 32 mg PBN / kg body weight to restore the young enzyme levels compared to A, B and C
It is as effective as administering kg body weight. Appropriate dosages and the full range of effective dosages for other species of animals can be determined by similar methodology.

10 mg PBN / kg (A, B and C in FIG. 12)
Alternatively, these figures show the time-related changes in protein oxidation and enzyme activity after cessation of twice daily dosing with 32 mg PBN / kg (FIGS. 13A, B and C). The results indicate that although the half-life of PBN itself is 3 hours, the effect of PBN is unchanged from 1 to 3 days after the end of the twice daily treatment with PBN. At day 7, enzyme levels have changed by about 50%. At day 14, oxidase has returned to near pre-treatment levels.

Example 6: Demonstration of a correlation between the effect of PBN on brain enzymes and memory Figure 14 shows that there is a functional reflection for such treatment. Young and old gerbils were tested for spatial and short-term memory in an 8-arm radial maze. Put the animal in the center of the 8-arm radial maze,
All eight arms of the radial maze were searched. Upon completing the test exploring each of the eight arms, the animals were removed from the maze. The number of times the animal re-entered the previously entered arm was counted as an error. Under ideal conditions, the animal enters each of the eight arms once, but does not re-enter any arm. Young adult gerbils (young) often penetrated each arm without re-entering any arm. Furthermore, although the time required to penetrate all arms was recorded, its effectiveness for short-term memory function cannot be confirmed. FIG.
As can be seen from Fig. 4, untreated young gerbils
There were fewer errors during testing than untreated aged gerbils.

[0164] Although exposed to transient ischemia for a period of time, which is also an oxidative process, young adult gerbils made an average of 15 substantial errors in such tests. In the period after the ischemic period, there is significant carbonyl protein formation and reduced glutamine synthetase activity similar to those seen in senescence.

Treatment of gerbils for 7 days resulted in a significant change in the number of errors observed in aged gerbils.
Young gerbils given PBN for 7 days did not differ from control gerbils. Meanwhile, PBN is 7
Aged gerbils fed for days showed a marked reduction in the number of errors and a return to the range of behavior exhibited by control young gerbils. Thus, there is a functional reflection corresponding to the biochemical changes seen in decreased protein carbonyl and increased glutamine synthetase activity after long-term PBN treatment. This functional reflection is indicated by the number of errors found in the short-term spatial memory in the radial arm maze. Note that the radial arm maze test does not require a food boost or other compensation to accompany the test. An unknown animal is placed in a radial arm maze and tested once. And the difference in the test is reliable even if retested.

[0166]

The present invention provides compounds, compositions and methods of using them for treating or preventing dysfunctions and disease states resulting from oxidative damage.

[Brief description of the drawings]

FIG. 1. PBN against transient ischemia-induced hyperactivity
FIG. 4 shows a graph of a dose-effect curve for the beneficial effects of.

FIG. 2 is a graph showing the effect of PBN on the level of activity activity.

FIG. 3 is a graph showing the effect of PBN on activity activity after ischemia.

FIG. 4 is a graph showing the effect of PBN on changes in voluntary behavioral activity.

FIG. 5 is a graph showing the effect of PBN on behavioral activity.

FIG. 6 is a graph showing the effect of PBN on the survival rate after performing lethal active occlusion of the carotid artery.

FIG. 7: For control of carbonyl protein
It is a figure showing the graph showing the effect of PBN.

FIG. 8 is a graph showing the effect of PBN on glutamine synthetase control.

FIG. 9 is a graph showing a correlation between carbonyl / mg protein and glutamine synthetase specific activity.

FIG. 10. PBN against cortical alkaline protease activity
It is a figure showing the effect of.

FIG. 11 shows the effect of PBN on protein and glutamine synthetase activity in the cerebral cortex.

FIG. 12 shows the effects of PBN on proteins, glutamine synthetase activity and protease activity in the cerebral cortex.

FIG. 13 shows the effects of PBN on proteins, glutamine synthetase activity, and protease activity in the cerebral cortex.

FIG. 14 shows the effect of PBN on radial maze experiments.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 25/28 A61P 25/28 25/30 25/30 39/06 39/06 43/00 105 43/00 105 (71) Applicant 594003676 825 N.C. E. FIG. 13th Street, Oklahoma City, Oklahoma 73104, United States of America (71) Applicant 591157305 University of Kentucky Research Foundation, Kentucky 40506, Lexington, United Kingdom, Canada ) Inventor John M. Carney United States Kentucky 50403, Lexington, Palomore 4033 (72) Inventor Robert A. Floyd USA Oklahoma 73120, Oklahoma City, Arrowhead Telas 12621 F-term (reference) 4C086 AA01 AA02 BC06 BC17 MA01 MA04 NA14 ZA02 ZA15 ZB21 ZC37 ZC39 4C206 AA01 AA02 HA08 MA01 MA04 NA14 ZA02 ZA15 ZB21 ZC37 Z

Claims (1)

[Claims] 1. A composition for the treatment or prevention of dysfunctions and disease states resulting from oxidative damage unrelated to bacterial toxins, comprising: a pharmaceutically acceptable carrier for topical administration to a patient; Comprising an active ingredient, wherein the active ingredient is represented by the following formula: Where X is phenyl or Where R is Or And n is an integer from 1 to 5; A t-butyl group in which one or more sites where Y can be hydroxylated or acetylated; phenyl; or Where W is: And Z is a C ₁ to C ₅ linear or branched alkyl group; or the active ingredient is represented by the following formula: Here, A and B are independently, -CH _3, -CH ₂ O
H, —CH ₂ OW or Where n is an integer from 1 to 5 and W is And Z is a C ₁ to C ₅ linear or branched alkyl group; or the active ingredient is represented by the following formula: Wherein Y is a t-butyl group at which one or more sites can be hydroxylated or acetylated; phenyl; or Where W is: And S is -H,-(OR) _n , where R is n is an integer from 1 to 4; And Z is a C ₁ to C ₅ linear or branched alkyl group.